Name: Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations (Video) | JoVE Journal
Uploaded: 2024-04-26T14:30:00.000+00:00
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Description: Discover in silico synthesis of carbonylated amino acids using molecular dynamics simulations to assess structural effects on proteins. This computational protocol enables researchers to study post-translational modifications and their impact on protein function.

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This work was supported by research grant code 1107-844-67943 from Ministerio de Ciencia, Tecnolog&#237;a e Innovaci&#243;n (Minciencias) and the University of Cartagena (Colombia) for grant to support the research groups 2021 and Acta 017-2022.

In Silico Structural Analysis of Protein Carbonylation by Lipid Derivatives

<ol><li>Cornell, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+second+generation+force+field+for+the+simulation+of+proteins%2C+nucleic+acids%2C+and+organic+molecules%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">A second generation force field for the simulation of proteins, nucleic acids, and organic molecules.</a> J Am Chem Soc. 117 (19), 5179-5197 (1995).</li>
<li>Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A., Case, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+testing+of+a+general+amber+force+field%5BTitle%5D)+AND+%22J+Comput+Chem%22%5BJournal%5D)">Development and testing of a general amber force field.</a> J Comput Chem. 25 (9), 1157-1174 (2004).</li>
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<li>Daura, X., Mark, A. E., van Gunsteren, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parametrization+of+aliphatic+CHn+united+atoms+of+GROMOS96+force+field%5BTitle%5D)+AND+%22J+Comput+Chem%22%5BJournal%5D)">Parametrization of aliphatic CHn united atoms of GROMOS96 force field.</a> J Comput Chem. 19 (5), 535-547 (1998).</li>
<li>Robertson, M. J., Tirado-Rives, J., Jorgensen, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+peptide+and+protein+torsional+energetics+with+the+OPLS-AA+force+field%5BTitle%5D)+AND+%22J+Chem+Theory+Comput%22%5BJournal%5D)">Improved peptide and protein torsional energetics with the OPLS-AA force field.</a> J Chem Theory Comput. 11 (7), 3499-3509 (2015).</li>
<li>Guvench, O., MacKerell, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+protein+force+fields+for+molecular+dynamics+simulations%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Comparison of protein force fields for molecular dynamics simulations.</a> Methods Mol Biol. 443, 63-88 (2008).</li>
<li>Petrov, D., Margreitter, C., Grandits, M., Oostenbrink, C., Zagrovic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+systematic+framework+for+molecular+dynamics+simulations+of+protein+post-translational+modifications%5BTitle%5D)+AND+%22PLoS+Comput+Biol%22%5BJournal%5D)">A systematic framework for molecular dynamics simulations of protein post-translational modifications.</a> PLoS Comput Biol. 9 (7), e1003154(2013).</li>
<li>Alviz-Amador, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+benchmark+to+obtain+AMBER+parameters+dataset+for+non-standard+amino+acids+modified+with+4-hydroxy-2-nonenal%5BTitle%5D)+AND+%22Data+Brief%22%5BJournal%5D)">Development and benchmark to obtain AMBER parameters dataset for non-standard amino acids modified with 4-hydroxy-2-nonenal.</a> Data Brief. 21, 2581-2589 (2018).</li>
<li>Pineda-Alemán, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cysteine+carbonylation+with+reactive+carbonyl+species+from+lipid+peroxidation+induce+local+structural+changes+on+thioredoxin+active+site%5BTitle%5D)+AND+%22J+Mol+Graph+Model%22%5BJournal%5D)">Cysteine carbonylation with reactive carbonyl species from lipid peroxidation induce local structural changes on thioredoxin active site.</a> J Mol Graph Model. 124, 108533(2023).</li>
<li>Alviz-Amador, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+4-HNE+modification+on+ZU5-ANK+domain+and+the+formation+of+their+complex+with+%CE%B2-Spectrin%3A+A+molecular+dynamics+simulation+study%5BTitle%5D)+AND+%22J+Chem+Info+Model%22%5BJournal%5D)">Effect of 4-HNE modification on ZU5-ANK domain and the formation of their complex with β-Spectrin: A molecular dynamics simulation study.</a> J Chem Info Model. 60 (2), 805-820 (2020).</li>
<li>Zhou, A., Schauperl, M., Nerenberg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Benchmarking+electronic+structure+methods+for+accurate+fixed-charge+electrostatic+models%5BTitle%5D)+AND+%22J+Chem+Info+Model%22%5BJournal%5D)">Benchmarking electronic structure methods for accurate fixed-charge electrostatic models.</a> J Chem Info Model. 60 (1), 249-258 (2020).</li>
<li>Gęgotek, A., Skrzydlewska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+effect+of+protein+modifications+by+lipid+peroxidation+products%5BTitle%5D)+AND+%22Che+Phys+Lipids%22%5BJournal%5D)">Biological effect of protein modifications by lipid peroxidation products.</a> Che Phys Lipids. 221, 46-52 (2019).</li>
<li>Moldogazieva, N. T., Zavadskiy, S. P., Astakhov, D. V., Terentiev, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipid+peroxidation%3A+Reactive+carbonyl+species%2C+protein%2FDNA+adducts%2C+and+signaling+switches+in+oxidative+stress+and+cancer%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Comm%22%5BJournal%5D)">Lipid peroxidation: Reactive carbonyl species, protein/DNA adducts, and signaling switches in oxidative stress and cancer.</a> Biochem Biophys Res Comm. 687, 149167(2023).</li>
</ol>

The authors have nothing to disclose.

One of the critical steps in developing the AMBER parameterization protocol was the quantum optimization of the new amino acid residues modified with the lipid peroxidation derivatives, due to the energetic variability related to the minimization and the way of assigning RESP charges in the AMBER antechamber. For this, ab initio optimization methods with Hartree-Fock (HF/6-31G) and semiempirical density functional theory (DFT; B3LYP/6-31G and M062X/6-31G) were established to evaluate the response to the load assignment. As a result, the HF functional presented better performance/computational cost ratio, taking this into account as a previous step to the protocol. This was also demonstrated in the study by Zhou et al.12.
During the application of the protocol there can be several sources of errors. The possible steric hindrances generated by the additional structures of the modification often lead to errors that are often solved through the minimization steps of the molecular system. On the other hand, the parameters of the dihedral angles are usually adjusted at the end of the parameterization process and therefore, sometimes they tend to show as a possible error, in this particular case it is suggested to adjust the parameters by homology, as reported by Alviz-Amador9 and add it in the new format to eliminate the error.
One of the limitations of the method is the effort required for the step-by-step development of the parameterizations. The generation of new parameters from the new electronic structures and then adapting these parameters to protein systems requires a lot of dedication for its good execution. Therefore, a good strategy when implementing our protocol is to follow the step-by-step instructions and read the guide carefully.
In the landscape of molecular dynamics simulations, the significance of the AMBER protocol becomes evident. Its adaptive nature and versatility make it a valuable tool for researchers exploring diverse research areas. Beyond its application in protein systems, its extension to macromolecular structures opens doors to new possibilities. This adaptability not only addresses the existing gaps in standard parameterization methods but also offers a pathway for the creation of novel structures, thus expanding the horizons of molecular dynamics research. On the contrary, other investigations demonstrate that the conventional parameterization of post-translational modifications is limited to a particular modification type and is exclusively derived from publicly available repositories8, lacking the capability to generate novel structures.
Modifications resulting from the presence of reactive carbonyl species are frequently associated with a range of pathologies, including cancer, metabolic disorders, and degenerative diseases following different mechanisms13,14 . The support provided by this protocol is useful to assess various crucial properties, such as conformational stability, atomic flexibility, loss of secondary structures, solvent accessibility, and protein-protein interaction energy, among others. Consequently, the measurement of these properties could prove beneficial in situations where carbonylated proteins can induce irreversible alterations in biological systems, leading to conformational instability, increased or decreased atomic flexibility, and loss of secondary structure10,11.
In conclusion, the AMBER parameterization protocol, with its critical steps, adaptability, and versatility, stands as a pioneering method in the realm of molecular dynamics simulations. While acknowledging its limitations, its significance is underscored by its ability to address the shortcomings of existing methods, providing researchers with a powerful tool to explore the intricacies of molecular structures and behaviors across a spectrum of biological and chemical systems.

In the constant pursuit of understanding the molecular behavior of proteins with oxidative modifications, computational chemistry has become a fundamental pillar in the broad field of scientific research. This relies on the use of theoretical models capable of interpreting physical phenomena in electronic systems, using mathematical equations to describe the atomic behavior of molecules. Within this landscape, computational simulations of proteins stand out as crucial tools to analyze the atomic behavior of molecular systems. Based on the evaluation of structural behavior, energetic calculations, and conformational states1, these methods become strategic allies to predict the behavior of biomolecular systems.
These simulations specialize in studying structural changes and assessing the loss or gain of biological functions in protein systems. However, computational approaches have shown significant limitations when applied to protein systems containing modified residues formed by covalent post-translational modifications in the sequence. This is because many available methods lack resources with parameters adaptable to force fields that are compatible with the most common packages of programs for molecular dynamics simulations of proteins2,3,4,5,6. Therefore, the standardization of computational software-compatible force-field adaptive parameters is essential to facilitate the precise coupling of topologies and atomic coordinates with the equation governing the potential energy of the system7.
In response to these challenges, a protocol adaptable to new modified amino acid residues with aldehydes derived from lipid peroxidation has been developed using ab initio methods. In that sense, the optimization of the structural geometry of the new residues allows the assignment of adaptive charges to new bond, angle, and dihedral parameters that can be run in general force fields such as AMBER. Subsequent validation of these parameters allows the determination of the consistency and robustness of the method applicable to molecular dynamics simulations.
One of the notable strengths of this method lies in its ability to adapt to diverse post-translational modifications, from carbonylation to phosphorylation, acetylation, and methylation, among others. This versatility is not only limited to protein systems but extends to macromolecular structures, allowing coupling with atomic topologies and coordinates. In contrast, previous studies reveal that the standard parameterization of post-translational modifications is only suited to a specific type of modification and can only be obtained from published repositories, lacking the ability to create new structures8.
Currently, challenges in protein structure prediction and design are becoming more evident when modeling structures with post-translational modifications. The scarcity of parameters describing alterations at specific amino acid sites underlines the urgent need to develop and apply computational methods that can be adjusted to standard parameterizations. The aim of this protocol is to provide a route for the in silico construction of new parameters for amino acids covalently modified with reactive carbonyl species derived from fatty acid oxidation. These modified amino acids are recognized by the general amber force field (GAFF) and can, therefore, be used to evaluate in silico the structural and functional effects that this kind of carbonylation has on their target proteins.

<table><tbody><tr><td>AmberTools16 or Upper</td><td>The Amber Project</td><td></td><td>Amber is a suite of biomolecular simulation programs</td></tr><tr><td>Gaussian 09 or Upper</td><td>Gaussian Inc</td><td></td><td>Draw and optimize structures</td></tr><tr><td>Linux Ubuntu</td><td>GNU/Linux</td><td></td><td>Platform for AmberTools</td></tr><tr><td>NVIDIA GPUs GTX 1080 or Upper</td><td>Nvidia</td><td></td><td>Compatible with PMEMD</td></tr></tbody></table>

synthesizing amino acids modified with reactive carbonyls in silico to assess structural effects using molecular dynamics simulations

Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations

Protein carbonylation by reactive aldehydes derived from lipid peroxidation leads to cross-linking, oligomerization, and aggregation of proteins, causing intracellular damage, impaired cell functions, and, ultimately, cell death. It has been described in aging and several age-related chronic conditions. However, the basis of structural changes related to the loss of function in protein targets is still not well understood. Hence, a route to the in silico construction of new parameters for amino acids carbonylated with reactive carbonyl species derived from fatty acid oxidation is described. The Michael adducts for Cys, His, and Lys with 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (HHE), and a furan ring form for 4-Oxo-2-nonenal (ONE), were built, while malondialdehyde (MDA) was directly attached to each residue. The protocol describes details for the construction, geometry optimization, assignment of charges, missing bonds, angles, dihedral angles parameters, and its validation for each modified residue structure. As a result, structural effects induced by the carbonylation with these lipid derivatives have been measured by molecular dynamics simulations on different protein systems such as the thioredoxin enzyme, bovine serum albumin and the membrane Zu-5-ankyrin domain employing root-mean-square deviation (RMSD), root mean square fluctuation (RMSF), structural secondary prediction (DSSP) and the solvent-accessible surface area analysis (SASA), among others.

To illustrate the implementation of the protocol and evaluate the results, the following analyses will be considered. The data set generated by assigning new parameters to modified amino acid residues was constructed based on optimization of the electronic structures, which were supported for partial RESP loadings. Figure 9 shows the structural conformation of one of the amino acid residues optimized with the parameter assignment.
<img alt="Molecular structure diagram, 3D chemical model with atoms labeled (e.g., C, O, N, S, H)." class="xfigimg" src="/files/ftp_upload/66605/66605fig09.jpg" data-altupdatedat="1788304762000" data-alt="Figure 9"> 
Figure 9: Cys-HHE residue synthesized in silico. Representation of HHE-modified cysteine amino acid with assigned topology and coordinate parameters. <a href="https://www.jove.com/files/ftp_upload/66605/66605fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The structures obtained from theoretical DFT levels with M062X/6-31G were compared with the classical mechanics structures through molecular dynamics simulations in AMBER. Each of the parameters obtained from the simulations showed a good correlation with the theoretical data from quantum mechanics. The average bond distance errors showed values of approximately 0.001 - 0.002 Å, while the angles were ~ 8.2°. The typology, distances and constants of bonds and angles are listed in Table 1. These data were similar to those reported in the data article by Alviz-Amador et al.9. Parameter files are available at http://research.bmh.manchester.ac.uk/bryce/amber/.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="7">Cys-HHE</td>
		</tr>
		<tr>
			<td rowspan="2">Methods</td>
			<td colspan="3">Bond </td>
			<td colspan="3">Angle</td>
		</tr>
		<tr>
			<td colspan="3">(Å, ± Stdev)</td>
			<td colspan="3"> (°, ± Stdev)</td>
		</tr>
		<tr>
			<td>QM</td>
			<td>S1 –C4</td>
			<td>C6-C8</td>
			<td>C8-C9</td>
			<td>S1-C4-C5</td>
			<td>O2-C6-C8</td>
			<td>C6-C8-C9</td>
		</tr>
		<tr>
			<td> (m062x/631g(d)</td>
			<td>1.82</td>
			<td>1.52</td>
			<td>1.53</td>
			<td>115.9</td>
			<td>109.25</td>
			<td>112.21</td>
		</tr>
		<tr>
			<td rowspan="2">MM (AMBER) aa alone</td>
			<td>1.85±</td>
			<td>1.55±</td>
			<td>1.54±</td>
			<td>111.66±</td>
			<td>109.77±</td>
			<td>113.16±</td>
		</tr>
		<tr>
			<td>0.002</td>
			<td>0.002</td>
			<td>0.002</td>
			<td>0.152</td>
			<td>0.14</td>
			<td>0.148</td>
		</tr>
	</tbody>
</table>
Table 1: Bond distance and angle parameters comparison. The values of bond distances and angle obtained by quantum (QM) and classical methods showed no significant differences.
Once each of the parameters for the modified amino acid residues was generated and validated, the dynamic behaviors were examined through molecular dynamics simulations with trajectories of 1 µs in order to evaluate the effect on the stability of each residue compared to its native counterpart (Figure 10). The RMSD values obtained for each of the modified amino acids did not show significant differences from their native counterpart, and they maintained their conformational stability throughout the entire trajectory.
<img alt="Protein dynamics analysis, RMSD vs time graphs comparing oxidized and native cysteine stability." class="xfigimg" src="/files/ftp_upload/66605/66605fig10.jpg" data-altupdatedat="1788304762000" data-alt="Figure 10"> 
Figure 10: RMSD graph of synthesized residues in silico. Representative RMSD of unmodified and modified cysteine residue with HHE, HNE, MDA and ONE. <a href="https://www.jove.com/files/ftp_upload/66605/66605fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The files resulting from the parameterization of modified amino acid residues have been utilized to substitute natural structural amino acids in proteins that have experimental evidence of carbonylation. This substitution was undertaken to evaluate the structural and functional impacts that may occur in the protein as a result of these modifications. It had been reported by in silico studies of carbonylation by reactive carbonyl species on protein systems like Ankyrin and Thioredoxin10,11.

Here, we describe a protocol for the optimization and parameterization of amino acid residues modified with reactive carbonyl species, adaptable to protein systems. The protocol steps include structure design and optimization, charge assignments, parameter construction, and preparation of protein systems.

Protein carbonylation by reactive aldehydes derived from lipid peroxidation leads to cross-linking, oligomerization, and aggregation of proteins, causing ...

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Discover in silico synthesis of carbonylated amino acids using molecular dynamics simulations to assess structural effects on proteins. This computational protocol enables researchers to study post-translational modifications and their impact on protein function.

Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations (Video) | JoVE Journal

Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations

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